Microfluidics is a multidisciplinary field intersecting engineering, physics, chemistry, biochemistry, nanotechnology, and biotechnology, with practical applications to the design of systems in which low volumes of fluids are processed to achieve multiplexing, automation, and high-throughput screening. Advances in microfluidics technology are revolutionizing molecular biology procedures for enzymatic analysis (e.g., glucose and lactate assays), DNA analysis (e.g., polymerase chain reaction and high-throughput sequencing), and proteomics. The basic idea of microfluidic biochips is to integrate assay operations such as detection, as well as sample pre-treatment and sample preparation on one chip.
There is increased evidence that phenotypic and genotypic heterogeneity in cell populations widely exists. The key information from individual rare cells may be masked by bulk cell analysis. Single-cell analysis, especially sequencing of DNA and RNA, has therefore become significantly important for clonal mutation, tumor evolution, embryonic development, and immunological intervention.
The initial and key step for such downstream single-cell genetic analysis is to effectively isolate live single cells of interest from heterogeneous cell populations into submicroliter medium volume, followed by PCR (polymerase chain reaction) analysis. (PCR is a technology in molecular biology used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence.)
Besides laser capture microdissection primarily used to isolate single cells from formalin-fixed paraffin-embedded tissue, there are currently four main approaches for single-cell isolation from cell suspensions. The most frequently used method is serial dilution, which has been widely applied for colony formation but is not suited for PCR analysis where single cells should be isolated into submicroliter suspensions for a better amplification reaction. Even so, these colonies are still required for further analysis to judge if they originate from single cells which are very difficult to be accurately counted after seeding into 96-well plates. A second method is micromanipulation, which is mainly developed to isolate single cells for genome/transcriptome sequencing. However, micromanipulation is a time-consuming process and its low throughput feature makes it difficult to rapidly prepare dozens and even hundreds of single cells. Moreover, it is highly dependent on a researcher's ability to suck single cells. Another method is flow cytometry, which is a well-established method particularly suitable for high-throughput sorting of specific cells based on a preset fluorescence gating strategy. However, maintenance of high single-cell viability is challenging and it doesn't work when only a limited number of cells, such as precious clinical samples, are available.
Due to the comparable dimensions of microchannels and cells, microfluidic technology provides a unique and efficient method for single cell manipulation. However, potential disadvantages, including requirement of additional skills for microfluid manipulation, poor compatibility with existing experimental platforms, and inability/difficulty to selectively retrieve the isolated single cells from microchips for further analysis, greatly limit its application in common laboratories. The original Microfluidic Aliquot Chip (MA-Chip) comprises 120 channels that directly connect to one center inlet well in a radial pattern, resulting in a space of less than 40 μm between two neighboring channels around the center inlet well. Although the 40 μm of fabrication resolution can be well achieved by photolithography to make PDMS MA-Chips, it is challenging to fabricate such MA-Chips with plastic materials, such as polystyrene (PS), polypropylene (PP), polycarbonate (PC), and polymethyl methacrylate (PMMA), by using injection molding or laser cutting. Therefore, an alternative design of MA-Chip with the increased space of two neighboring channels is required for the mass fabrication of plastic MA-Chips.
The branched MA-Chip Type 1 (bMA-Chip T1) in the present invention is designed to increase the space between two neighboring channels around the center inlet well while maintaining uniform liquid distribution from the center inlet well to the outlet wells. The original MA-Chip contains one segment, in contrast, the bMA-Chip T1 contains multiple segments, allowing the channel number around the center inlet well to decrease from 120 to 24 and even 12. These improvements reduce channel density and provide an extra space around the center inlet well. As a result, the space between neighboring channels increases from less than 40 μm to more than 400 μm. The improved design in bMA-Chip T1 can meet the requirement for the mass fabrication of plastic MA-Chips.
An objective of the present invention is to provide a technique for simple, rapid, and versatile single-cell isolation using microfluidic technology. In this invention, a single cell is isolated by aliquoting from a suspension of a large number of cells, independently of cell size, shape, and motility. The original microfluidic aliquot chip provides such functions, however it consists of a plurality of straight channels in the chip, resulting in the densely positioned channels. The branched Microfluidic Aliquot Chip-Type 2 (bMA Chip-T2) and the branched Microfluidic Aliquot Chip-Type 3 (bMA Chip-T3) in the present invention are designed as an improvement to the original MA-chip due to the multiple branched channels. This offers the advantage of reduced clogging, strengthened sealing, and enhanced isolation through uniform distribution of flow resistance into the branched channels.
The original design and fabrication methods of the original MA-Chip are based on photolithography followed by soft PDMS casting on the mold. While the photolithography based manufacturing process ensures high resolution of the microstructures in MA-Chip, the daily manufacturing output is limited. The roughly estimated production cycle time for a MA-Chip is 1 hr. This low production rate results in high production costs. To reduce both production costs and final market price, the MA-Chip must be redesigned so that it is appropriate for standard mass production strategy such as the injection molding process. The new design is suitable for a high output production process such as injection molding to greatly increase the production rate.
The injection mold design of the present MA-Chip allows mass production by the injection molding process. With standard operation, the estimated production cycle time for a single device is 10 s, which is 360 times higher than the original rate. Compared to the original design, the new design also improves MA-Chip function, operation, and compatibilities. The device is assembled and packaged for ready to use application. It reduces additional operations such as placing the MA-Chip on a flat sterile surface and ensuring sealing of the flow channel.
The original MA-Chip has a unique design of radial channels connecting a center inlet to surrounding outlet wells and provides the capability to isolate and identify rare single cells in a mixed population with a simple pipetting operation. The vital design and fabrication element is the smooth connection of micrometer scale channels with millimeter scale wells. In the original manufacturing scheme, the micrometer size channels (30-80 μm) are fabricated by soft lithography followed by PDMS molding, and the millimeter scale wells (1.5-2 mm) are created by mechanical punch press. Thus, this manual operation demands a significant amount of time and labor. To improve throughput of the hole punch process, a multiplexed hole punch device is designed.
A multiplex hole punch strategy for the rapid fabrication of MA-Chip is designed to meet the requirement of mass production while maintaining the original MA-Chip manufacturing format. Current operation requires the holes to be punched manually by trained individuals. In one MA-Chip, there are 96-120 holes and alignment is required in each hole punch process. The quality of outcome and labor time in this process highly depends on the operator's skill and experience. The multiplex hole punch is designed to increase throughput of the hole punch process while maintaining the original design format of the MA-Chip.
Photolithography is suitable for fabricating high quality PDMS channels but difficult for making holes. In contrast, laser cutting or injection molding can easily achieve mass production of plastic holes but difficult to make high quality channels. The two methods can be combined to achieve rapid fabrication of MA-Chips. The present invention includes a basic strategy for the rapid fabrication of MA-Chip to meet the requirement of mass production. The operation is to align and combine two patterned layers.
In the original MA-Chip, the outlet wells are primarily located in the edge of the device with a radial pattern. However, the majority of the MA-Chip is occupied by radial channels, resulting in wasted space and difficulty in further increasing the number of outlet wells to meet the requirement of high-throughput assay, such as a device containing hundreds to thousands of wells. Therefore, a new design of the MA-Chip is required. The present invention has a rectangular MA-Chip (rMA-Chip) that has the potential to integrate hundreds to thousands of outlet wells in the size of a standard 96-well plate.
U.S. Pat. No. 6,632,656 (Oct. 14, 2003; Thomas et al.), incorporated by reference herein, discloses apparatus and methods for performing cell growth and cell based assays in a liquid medium. The apparatus comprises a base plate supporting a plurality of micro-channel elements, each micro-channel element comprising a cell growth chamber, an inlet channel for supplying liquid sample thereto and an outlet channel for removal of liquid sample therefrom, a cover plate positioned over the base plate to define the chambers and connecting channels, the cover plate being supplied with holes to provide access to the channels. Means are incorporated in the cell growth chambers, for cell attachment and cell growth. More particularly, as shown and described therein:                Referring to FIG. 1b, the apparatus comprises a rotatable disc (18) microfabricated to provide a sample introduction port located towards the centre of the disc and connected to an annular sample reservoir (9) which in turn is connected to a plurality of radially dispersed micro-channel assay elements (6) each of said micro-channel elements comprising a cell growth chamber, a sample inlet channel and an outlet channel for removal of liquid therefrom and a cover plate positioned onto said disc so as to define closed chambers and connecting channels. Each micro-channel element is connected at one end to the central sample reservoir (9) and at the opposing end to a common waste channel (10).        Each of the radially-dispersed micro-channel elements (6) of the microfabricated apparatus (shown in FIG. 1a) comprises a sample inlet channel (1) connected at its left hand-end end to the reservoir (9), a cell growth chamber (2) for performing cell growth and connected through a channel (4) to an assay chamber (3) and an outlet channel (5) connected at its right-hand end to the waste channel (10).        Suitably the disc (18) is of a one- or two-piece moulded construction and is formed of an optionally transparent plastic or polymeric material by means of separate mouldings which are assembled together to provide a closed structure with openings at defined positions to allow loading of the device with liquids and removal of waste liquids. In the simplest form, the device is produced as two complementary parts, one or each carrying moulded structures which, when affixed together, form a series of interconnected micro-channel elements within the body of a solid disc. Alternatively the micro-channel elements may be formed by micro-machining methods in which the micro-channels and chambers forming the micro-channel elements are micro-machined into the surface of a disc, and a cover plate, for example a plastic film, is adhered to the surface so as to enclose the channels and chambers.        The scale of the device will to a certain extent be dictated by its use, that is the device will be of a size which is compatible with use with eukaryotic cells. This will impose a lower limit on any channel designed to allow movement of cells and will determine the size of cell containment or growth areas according to the number of cells present in each assay. An average mammalian cell growing as an adherent culture has an area of ˜300 μm2; non-adherent cells and non-attached adherent cells have a spherical diameter of ˜10 μm. Consequently channels for movement of cells within the device are likely to have dimensions of the order of 20-30 μm or greater. Sizes of cell holding areas will depend on the number of cells required to carry out an assay (the number being determined both by sensitivity and statistical requirements). It is envisaged that a typical assay would require a minimum of 500-1000 cells which for adherent cells would require structures of 150,000-300,000 μm2, i.e. circular ‘wells’ of ˜400-600 μm diameter.        The configuration of the micro-channels . . . is preferably chosen to allow simultaneous seeding of the cell growth chamber by application of a suspension of cells in a fluid medium to the sample reservoir by means of the sample inlet port, followed by rotation of the disc (18) by suitable means at a speed sufficient to cause movement of the cell suspension outward towards the periphery of the disc by centrifugal force. The movement of liquid distributes the cell suspension along each of the inlet micro-channels (1, 8) towards the cell growth chambers (2, 7). The rotation speed of the disc is chosen provide sufficient centrifugal force to allow liquid to flow to fill the cell growth chamber (2, 7), but with insufficient force for liquid to enter the restricted channel (4, 16) of smaller diameter on the opposing side of the cell growth chamber.        